CAREER: Investigation of DNA-binding Protein Dynamics With High-resolution Optical Traps

一大类DNA结合蛋白以一种非序列特异性的方式与基因组相互作用。这些蛋白质在底物上以机械方式作用，通过弯曲、扭曲或拉伸分子改变DNA构象，并通过寡聚形成长丝。这些核蛋白复合体通常作为基因组维持过程发生的底物。因此，它们参与DNA新陈代谢的各个方面，包括复制、重组和修复，并且是细胞过程的重要调节因子。单链DNA结合蛋白(SSB)作为这类蛋白质共同特征的模型系统。这个项目将使用传统生物化学和分子生物学的综合技术，结合单分子生物物理学和计算生物学来研究：(1)SSB如何诱导核酸的构象重排，(2)它们如何齐聚成核蛋白细丝，以及(3)这些蛋白质簇如何招募和调节参与核酸加工的其他蛋白质的活性。具体地说，高分辨率光学捕获与单分子荧光技术相结合将被用来揭示蛋白质-DNA的动态相互作用，超越了目前方法的限制。这项工作将阐明基因组维护的基本方面。广泛的影响：PI对年轻科学家的跨学科教育和面向代表性不足的群体有着深切的承诺。特别是，他在生物物理学中看到了一个独特的机会，可以招募传统上被生物学而不是物理学吸引的女性进入定量科学。该项目的外展和教育部分将这些主题综合为一个针对初中、高中、本科生和研究生教育的广泛计划。具体地说，PI将：(1)为女孩暑期计划建立一个实验营，向初中和高中女孩传授物理学及其对生物学问题的影响，并提供生物物理学的实践经验；(2)改进面向生命科学专业学生(主要是女性)的本科生物理入门课程的教学，以便更好地将物理概念与生物学和医学联系起来；(3)作为NSF活细胞物理中心(CPLC)的一部分，开发一个致力于培训先进技术下一代生物物理学家的实验室课程，以及(4)参加每年一次的少数族裔会议。该项目由分子和细胞生物科学部的基因和基因组系统集群和生物分子系统集群共同支持。

A broad class of DNA-binding protein interacts with the genome in a non-sequence-specific manner. These proteins act mechanically on their substrates, altering DNA conformation by bending, twisting, or stretching the molecule, and oligomerizing to form long filaments. These nucleoprotein complexes often serve as substrates upon which genome maintenance processes occur. Thus, they are involved in all aspects of DNA metabolism, in replication, recombination, and repair, and are important regulators of cellular processes. Single-stranded DNA binding proteins (SSB) serve as a model system for features common to this class of proteins. This project will use a synthesis of techniques from traditional biochemistry and molecular biology, in combination with single-molecule biophysics and computational biology to investigate: (1) how SSBs induce conformational rearrangement of nucleic acids, (2) how they oligomerize into nucleoprotein filaments, and (3) how these protein clusters recruit and modulate the activity of other proteins involved in nucleic acid processing. Specifically, high-resolution optical trapping in combination with single-molecule fluorescence techniques will be used to reveal dynamic protein-DNA interactions, going beyond the limitations of current methods. This work will shed light on fundamental aspects of genome maintenance.Broader impact: The PI has a deep commitment to interdisciplinary education of young scientists and outreach towards underrepresented groups. In particular, he sees in biophysics a unique opportunity to recruit women, who have traditionally been drawn to biology over physics, into the quantitative sciences. The outreach and education components of the project synthesize these themes into a broad plan targeting middle and high school, undergraduate, and graduate education. Specifically, the PI will: (1) develop a lab camp for a girls' summer program to teach middle and high school girls about physics and its impact on biological problems and to provide hands-on experience with biophysics, (2) improve the teaching of an introductory undergraduate physics course for life science students (the majority of whom are women) to better connect physics concepts with biology and medicine, (3) develop a lab course devoted to the training of the next generation of biophysicists in advanced technologies as part of the NSF Center for the Physics of the Living Cells (CPLC), and (4) participate in yearly minority conferences. This project is jointly supported by the Genes and Genome Systems Cluster and the Biomolecular Systems Cluster in the Division of Molecular and Cellular Biosciences.